Mtb-Timer: a fluorescent reporter to visualize <i>Mycobacterium tuberculosis</i> replication and antibiotic responses

ABSTRACT Despite the importance of monitoring Mycobacterium tuberculosis (Mtb) replication and antibiotic responses at the single-cell level, available tools to accurately monitor single Mtb replication are limited. Here, we report the development of Mtb-Timer, a fluorescent reporter in Mtb that exploits the intrinsic green-to-red photoconversion of the Timer protein as a proxy for Mtb replication. We generated an Mtb H37Rv strain expressing Timer (Timer Mtb ) and monitored in vitro Mtb replication at the single-cell level by imaging and flow cytometry. Moreover, we show that exposure to the first-line anti-tuberculosis antibiotics isoniazid, rifampicin, ethambutol, and pyrazinamide resulted in a dose-dependent green-to-red photoconversion by imaging and flow cytometry. When we tested Mtb-Timer in cellulo using primary human monocyte-derived macrophages, we observed that intracellular Mtb populations displayed a striking phenotypic heterogeneity. Single-cell Mtb-Timer green-to-red ratios analyzed by both fixed and live imaging revealed intracellular Mtb populations with high green-to-red ratios. In contrast, treatment with antibiotics showed a decrease in green-to-red ratios in a dose-dependent manner by imaging and flow cytometry. Collectively, these results establish Mtb-Timer as a robust tool for monitoring Mtb replication and antibiotic responses, both in vitro and in cellulo , by imaging and flow cytometry. IMPORTANCE Tuberculosis treatment is challenging in part because Mycobacterium tuberculosis (Mtb) populations are phenotypically heterogeneous. Within the same infection, bacterial cells can exist in different physiological states, from actively replicating to slow-growing or non-replicating forms. To better study these processes, we developed and validated Mtb-Timer, a fluorescent reporter that enables visualization of bacterial growth dynamics. The system is based on a fluorescent protein that changes color over time: newly synthesized protein emits green fluorescence (GFP) and gradually shifts to red (DsRed) as it matures. The green-to-red fluorescence ratio, therefore, reflects bacterial replication activity. Using microscopy and flow cytometry in both in vitro cultures and infected macrophages ( in cellulo ), Mtb-Timer enables the quantification of distinct bacterial subpopulations and the real-time monitoring of the green-to-red fluorescence ratio and bacterial burden during treatment with first-line antibiotics.